The overall aim of our research is to develop a combined engineering, ultrastructural and biophysical approach to the mechanisms whereby endothelial cells and the clefts between the cells modulate microvessel permeability. During the current grant period we completed novel experiments guided by new three-dimensional theoretical models to relate the permeability properties of segments of individually perfused microvessels to the ultrastructure of the junctional strands between adjacent endothelial cells and fiber matrix components within-the cleft or at the endothelial surface. The primary focus has been the analysis of the three dimensional spread of low molecular weight tracer molecules on the abluminal side of the junction strand to determine the size and frequency of the pores in this strand. Because the wakes are much larger than pores, this new approach offers the possibility of detecting junction strand interruptions and small pores that lie beyond the resolution of conventional transmission electron microscopy. These studies have resulted in major revision of the current ideas about pathways for water and solute through junctional strands. Three Specific Aims are proposed to investigate new or revised themes in this proposal. The hypotheses to be tested under Specific Aim 1 are: (1) that the visible wakes formed by small electron-dense tracers on the abluminal side of the junction strand after short time perfusions are formed at widely separated discontinuities in the junctional strand; and (2) that the visible wakes on the abluminal side of the junctional strand after longer time perfusions are formed by diffusion through a population of very small pores distributed along the length of the strand. This wake is not detected until later times when the tracer concentration in the tissue has increased to a level close to a detection threshold. The hypothesis in Specific Aim 2 is that molecular sieving of larger molecular weight tracers is confined to the thin fiber matrix layer at the entrance region of the cleft. The hypothesis in Specific Aim 3 is that changes in permeability which are not due to the formation of gaps between adjacent endothelial cells in venular capillaries are the result of changes in the size and frequency of the discontinuities in the junctional strand and the structure of the molecular sieve at the luminal surface. Our approach provides new methods to investigate such subtle changes in junctional and matrix structure. In the proposed studies, theoretical modeling of water and solute transport through the interendothelial cleft and adjacent tissue will be developed further to interpret the time dependent wake experiments proposed in Specific Aims 1 and 3, and to analyze the results of the experiments with larger solute molecules proposed in Specific Aims 2 and 3. All experiments will be performed on individual perfused microvessels of precisely known permeability properties using microperfusion techniques and novel confocal methods to visualize tracer distribution around perfused microvessels. This combined theoretical and experimental approach is the most direct- approach to a new understanding of the nature of the junction and fiber matrix structures which modulate microvessel permeability.